Method enabling use of extracellular RNA extracted from plasma or serum to detect, monitor or evaluate cancer

ABSTRACT

This invention relates to the use of tumor-derived or associated extracellular ribonucleic acid (RNA) found circulating in the plasma or serum fraction of blood for the detection, monitoring, or evaluation of cancer or premalignant conditions. Extracellular RNA may circulate as non-bound RNA, protein-bound RNA, lipid-RNA complexes, lipoprotein (proteolipid)—RNA complexes, protein-RNA complexes including within or in association with ribonucleoprotein complexes, nucleosomes, or within apoptotic bodies. Any intracellular RNA found in plasma or serum can additionally be detected by this invention. Specifically, this invention enables the extraction of circulating RNA from plasma or serum and utilizes nucleic acid amplification assays for the identification, detection, inference, monitoring, or evaluation of any neoplasm, benign, premalignant, or malignant, in humans or other animals, which might be associated with that RNA. Further, this invention allows the qualitative or quantitative detection of tumor-derived or associated extracellular RNA circulating in the plasma or serum of humans or animals with or without any prior knowledge of the presence of cancer or premalignant tissue.

This application is a continuation of U.S. Ser. No. 09/155,152, filedSep. 22, 1998, now U.S. Pat. No., 6,329,179, which is a national phaseapplication under 35 U.S.C. §371 of International Application, No.PCT/US97/03479, filed Mar. 14, 1997 and claiming the benefit of U.S.provisional application Ser. No. 60/014,730, filed March 26, 1996, thedisclosure of each of which is incorporated by reference herein.

BACKGROUND OF THE INVENTION

Ribonucleic acid (RNA) is essential to the processes which allowtranslation of the genetic code to form proteins necessary for allcellular functions, both in normal and neoplastic cells. While thegenetic code structurally exists as deoxyribonucleic acid (DNA), it isthe function of RNA, existing as the subtypes transfer-RNA,messenger-RNA or messenger-like RNA, and ribosomal-RNA, to carry andtranslate this code to the cellular sites of protein production. In thenucleus, this RNA may further exist as or in association withribonucleoproteins (RNP). The pathogenesis and regulation of cancer isdependent upon RNA-mediated translation of specific genetic codes, whichoften reflects mutational events within oncogenes, to produce proteinsinvolved with cell proliferation, regulation, and death. Furthermore,other RNA and their translated proteins, although not necessarily thoseinvolved in neoplastic pathogenesis or regulation, may serve todelineate recognizable characteristics of particular neoplasms by eitherbeing elevated or inappropriately expressed. Thus, recognition ofspecific RNA can enable the identification, detection, inference,monitoring, or evaluation of any neoplasm, benign, malignant, orpremalignant, in humans and animals. Furthermore, since RNA can berepetitively created from its DNA template, for a given gene within acell there may be formed a substantially greater number of associatedRNA molecules than DNA molecules. Thus, an RNA-based assay should havegreater sensitivity, and greater clinical utility, than its respectiveDNA-based assay. Note that the term RNA denotes ribonucleic acidincluding fragments of ribonucleic acid consisting of ribonucleic acidsequences.

RNA based nucleic acid amplification assays, including the reversetranscriptase polymerase chain reaction (RT-PCR, also known as reversetranscription polymerase chain reaction or RNA-PCR), branched DNA signalamplification, and self-sustained sequence replication assays, such asisothermal nucleic acid sequence based amplification (NASBA), haveproven to be highly sensitive and specific methods for detecting smallnumbers of RNA molecules. As such, they can be used in direct assays ofneoplastic tissue (1-3). Since peripheral blood is readily obtainablefrom patients with cancer, and metastatic cancer cells are known tocirculate in the blood of patients with advanced cancer, severalinvestigators have recently used RT-PCR to detect intracellular RNAextracted from circulating cancer cells (4-7). It must be emphasizedthat currently investigators apply RT-PCR to detect extractedintracellular RNA from a predominately cellular fraction of blood inorder to demonstrate the existence of circulating cancer cells. RT-PCRis applied only to the cellular fraction of blood obtained from cancerpatients, i.e., the cell pellet or cells within whole blood. The plasmaor serum fraction of blood is usually discarded prior to analysis, butis not examined separately. Since such a cellular fraction approachrelies upon the presence of metastatic circulating cancer cells, it isof limited clinical use in patients with early cancers, and is notuseful in the detection of non-invasive neoplasms or pre-malignantstates.

The invention described by this patent application demonstrates thenovel use of that human or animal tumor-derived or tumor-associated RNAfound circulating in the plasma or serum fraction of blood, as a meansto detect, monitor, or evaluate cancer and premalignant states. Thisinvention is based upon the application of RNA extraction techniques andnucleic acid amplification assays to detect tumor-derived or associatedextracellular RNA found circulating in plasma or serum. In contrast tothe detection of viral-related RNA in plasma or serum, and the detectionof tumor-associated DNA in plasma and serum, the detection of human ormammalian RNA, and particularly tumor-derived or associated RNA, hasnever been detected specifically within the plasma or serum fraction ofblood using nucleic acid amplification methodology, and thus representsa novel and non-obvious use for these RNA extraction methods and nucleicacid amplification assays. Since this invention is not dependent uponthe presence of circulating cancer cells, it is clinically applicable tocases of early cancer, noninvasive cancers, and premalignant states, inaddition to cases of invasive cancer and advanced cancer. Further, thisinvention allows the detection of RNA in previously frozen or otherwisestored plasma and serum, thus making plasma and serum banks availablefor analysis and otherwise increasing general usefulness.

Tumor-derived or tumor-associated RNA that is present in plasma andserum may exist in two forms. The first being extracellular RNA, but thesecond being extractable intracellular RNA from cells occasionallycontaminating the plasma or serum fraction. In practice, it is notnecessary to differentiate between intracellular and extracellular inorder to detect RNA in plasma or serum using the invention, and thisinvention can be used for detection of both. The potential uses oftumor-derived or associated extracellular RNA have not been obvious tothe scientific community, nor has the application of nucleic acidamplification assays to detect tumor-derived or associated extracellularRNA been obvious. Indeed, the very existence of tumor-derived orassociated extracellular RNA has not been obvious to the scientificcommunity, and is generally considered not to exist. It is generallybelieved that plasma ribonucleases rapidly degrade any extracellularmammalian RNA which might circulate in blood, rendering it nondetectable(8). Komeda et al., for example, specifically added free RNA to wholeblood obtained from normal volunteers, but were unable to detect thatRNA using PCR (54). However, nucleases appear inhibited in the plasma ofcancer patients (9). In addition, extracellular RNA, either complexed tolipids and proteolipids, protein-bound, or within apoptotic bodies,would be protected from ribonucleases. Thus, although still undefined,tumor-derived or associated extracellular RNA may be present in plasmaor serum via several mechanisms. Extracellular RNA could be secreted orshed from tumor in the form of lipoprotein (proteo-lipid)-RNA orlipid-RNA complexes, it could be found within circulating apoptoticbodies derived from apoptotic tumor cells, it could be found inproteo-RNA complexes released from viable or dying cells including or inassociation with ribonucleoproteins, or in association with otherproteins such as galectin-3, or RNA could be released from necroticcells and then circulate bound to proteins normally present in plasma.Additionally it could exist circulating within RNA-DNA complexesincluding those associated with ribonucleoproteins and other nucleicRNA. Further, RNA may exist within several of these moietiessimultaneously. For example, RNA may be found associated withribonucleoprotein found within proteo-lipid apoptotic bodies. Thepresence of extracellular RNA in plasma or serum makes their detectionby nucleic acid amplification assays feasible.

Several studies in the literature support the existence of tumor-derivedor associated extracellular RNA. RNA has been shown to be present on thecell surface of tumor cells, as demonstrated by electrophoresis (10),membrane preparations (11), and P³² release (12). Shedding ofphospholipid vesicles from tumor cells is a well described phenomena(13, 14), and similar vesicles have been shown to circulate in the bloodof patients with cancer (15). Kamm and Smith used a fluorometric methodto quantitate RNA concentrations in the plasma of healthy individuals(55). Rosi and colleagues used high resolution nuclear magneticresonance (NMR) spectroscopy to demonstrate RNA molecules complexed withlipid vesicles which were shed from a human colon adenocarcinoma cellline (16). Further characterization of these lipid-RNA complexesdemonstrated the vesicles additionally contained triglycerides,cholesterol esters, lipids, oligopeptide, and phospholipids (17).Mountford et al. used magnetic resonance spectroscopy to identify aproteolipid in the plasma of a patient with an ovarian neoplasm (18).While further evaluation of the proteolipid using the orcinol methodsuggested RNA was present, this could not be confirmed using othermethods. Wieczorek and associates, using UV spectrometry and hydrolysisby RNases, claimed to have found a specific RNA-proteolipid complex inthe serum of cancer patients which was not present in healthyindividuals (19-20). The complex had unvarying composition regardless ofthe type cancer. Wieczorek et al. were further able to detect thisspecific RNA-proteolipid complex using a phage DNA cloned into E. coliand hybridized to RNA from neoplastic serum, a method distinctlydifferent from the method of this invention. The DNA was then detectedby immunoassay (21). However, the RNA found in this complex is describedas 10 kilobases, which is so large as to make it questionable whetherthis truly represents RNA as described. More recently, DNA andRNA-containing nucleoprotein complexes, possibly representing functionalnuclear suborganellular elements, were isolated from the nuclei oflymphoma cells (22). It was not shown, however, that these complexes canbe shed extracellularly. Other ribonucleoprotein complexes have beenassociated with c-myc oncogene RNA (56).

While plasma and serum are generally presumed to be cell-free, in thepractical sense, particularly under conditions of routine clinicalfractionation, plasma and serum may occasionally be contaminated bycells. These contaminating cells are a source of intracellular RNA whichis detectable by the methods of the invention. While the level ofcontaminating cells may be reduced by filters or high speedcentrifugation, these methods may also reduce extracellular RNA,particularly larger apoptotic bodies. Clinical utility of the inventionis not dependent upon further separating of plasma or serum RNA into itsextracellular and intracellular species.

While not related to the claims of this patent, similar analogy likelyexists for detection of normal RNA (non-tumor derived or non-tumorassociated RNA) in plasma and serum. Subsequent to the filing of theprovisional patent application for this patent, the inventor has shownthat normal RNA (non-tumor derived RNA) could similarly be detected inthe plasma or serum of both healthy volunteers and cancer patients usingextraction methods and amplification methods as described by thisinvention. Qualitative results suggested that amplified product wasgreater when obtained from cancer patients. Further, use of a 0.5 micronfilter prior to amplification reduced, but did not eliminate amplifiableRNA, consistent with extracellular RNA being of variable size, withadditional contaminating cells possible.

While the methods of RNA extraction utilized in this invention have beenpreviously used to extract both viral RNA and intracellular RNA, theirapplicability to extracellular tumor-related or tumor-associated RNA wasnot obvious. The physical characteristics of the extracellular RNAcomplexes remain largely unknown, and thus it was not known prior tothis invention if the methods of extraction to be described couldeffectively remove extracellular RNA from their proteo-lipid, apoptotic,vesicular, or protein-bound complexes. This invention describes theapplicability of these RNA extraction methods to the extraction ofextracellular RNA from plasma or serum, and thus describes a new use forthese extraction methods.

In summary, this invention describes a method by which RNA in plasma orserum can be detected and thus utilized for the detection, monitoring,or evaluation of cancer or premalignant conditions. This method utilizesnucleic acid amplification assays to detect human or animaltumor-derived or associated extracellular RNA circulating in plasma orserum. It also enables extraction and amplification of intracellular RNAshould cells be present in plasma or serum. The described extractionmethods and various nucleic acid amplification assays, including but notlimited to RT-PCR, branched DNA signal amplification, transciption-basedamplification, amplifiable RNA reporters, boomerang DNA amplification,strand displacement activation, cycling probe technology, isothermalNASBA amplification, and other self-sustained sequence replicationassays, have not been used for the detection of tumor-derived ortumor-associated RNA in plasma or serum, reflecting the generalscientific bias that mammalian extracellular RNA does not existcirculating in plasma or serum, despite isolated studies to thecontrary. Thus, this invention represents both a novel and non-obviousmethod of detecting, monitoring, and evaluating cancer or premalignantconditions, and a novel and non-obvious application of both RNAextraction methodology and nucleic acid amplification assays. Thisinvention, as described below entails a multi-step procedure applied toplasma or serum which consists of three parts, with the initial step(Part A) involving extraction of tumor-derived or associated RNA fromplasma or serum, a second step (Part B) involving application of anucleic acid amplification assay, in which reverse transcription of RNAto its cDNA may be involved, and a third step (Part C) involvingdetection of the amplified product. Any nucleic acid amplification assaycapable of permitting detection of small numbers of RNA molecules ortheir corresponding cDNA may be used in Part B. Similarly, variousmethods of detection of amplified product may be used in Part C,including but not limited to agarose gel electrophoresis, ELISAdetection methods, electrochemiluminescence, high performance liquidchromatography, and reverse dot blot methods. Furthermore, Part B andPart C may utilize assays which enable either qualitative orquantitative RNA analysis. Thus, while this invention uses variousmethods described in the literature, it is the unique application ofthese methods to the detection of tumor-derived or associatedextracellular RNA from plasma or serum that makes this invention novel.This invention provides a simple means for testing blood plasma or serumfor tumor-derived or associated RNA, with the result of identifyingpatients harboring tumor cells. Since this invention enables detectionof extracellular RNA, and does not depend upon the presence ofcirculating cancer cells, it offers a sensitive yet inexpensive screenfor both malignancy and pre-malignancy, as well as a way for monitoringcancer and obtaining other prognostically important clinicalinformation.

OBJECTS AND APPLICATIONS OF THE INVENTION

It is therefore the object of this invention to detect or infer thepresence of cancerous or precancerous cells whether from non-hematologicor hematologic malignancy, within a human or animal body, both in thoseknown to have cancer and in those not previously diagnosed, by examiningthe plasma or serum fraction of blood for tumor-derived or associatedextracellular RNA, including, but not limited to, that derived frommutated oncogenes, using nucleic acid amplification assays, such as, butnot limited to, polymerase chain reaction (RT-PCR), branched DNA signalamplification, isothermal nucleic acid sequence based amplification(NASBA), other self-sustained sequence replication assays,transcription-based amplification, boomerang DNA amplification, stranddisplacement activation, cycling probe technology, and amplifiable RNAreporters.

An application of this invention is to allow identification or analysis,either quantitatively or qualitatively, of tumor-derived or associatedRNA in the blood plasma or serum of humans or animals during orfollowing surgical procedures to remove premalignant or malignantlesions, and thus to allow stratification of such patients as to theirrisk of residual cancer following the surgery.

Another application of this invention is to allow identification oranalysis, either quantitatively or qualitatively, of tumor-derived orassociated RNA in the blood plasma or serum of humans or animals who arereceiving cancer therapies, including but not limited to biotherapy,chemotherapy, or radiotherapy, as a guide to whether adequatetherapeutic effect has been obtained or whether additional oralternative therapy is required, and further, to assess prognosis inthese patients.

Another application of this invention is to allow identification oranalysis, either quantitatively or qualitatively, of tumor-derived orassociated RNA in the blood plasma or serum of humans or animals whohave completed therapy as an early indicator of relapsed cancer,impending relapse, or treatment failure.

Another application of this invention is to allow identification, eitherby detection or by inference, of the presence of premalignant neoplasmsincluding dysplasias or adenomas by the examination of blood plasma orserum for RNA derived from or associated with those neoplasms.Furthermore, analysis, for example by a panel of assays to detectvarious RNA, may serve to distinguish malignant from premalignantconditions, or assist in medical monitoring to detect transformation ofa neoplasm to an outright malignancy, or to detect regression.

Thus, an application of this invention is to provide a method ofscreening both individuals without known risk, and individuals at risk,for cancer and premalignant conditions, and further, for defining riskof cancer when that risk is unknown.

Another application of this invention is to allow identification oranalysis, either quantitatively or qualitatively, of tumor-derived orassociated RNA in the blood plasma or serum of humans or animals eithernewly or recently diagnosed with cancer or a premalignant condition inorder to clarify when to initiate therapy, including adjuvant therapies.

Another application of this invention is to allow identification oranalysis of tumor-derived or associated RNA, either singularly or by apanel approach detecting varied RNA, in the blood plasma or serum ofhumans or animals in order to determine specific characteristics of agiven patient's tumor, as to assist in the development ofpatient-specific therapies, help direct a given patient into a giventreatment regimen, or help predict prognosis or tumor behavior.

SUMMARY OF THE INVENTION

The objects, advantages and applications of the present invention areachieved by the hereinafter described method for detecting tumor derivedor associated extracellular RNA from body fluids, in particular frommammalian blood plasma or serum by (A) extraction of RNA from bloodplasma or serum; (B) amplification of the RNA by nucleic acidamplification assays, including (1) reverse transcription polymerasechain reaction (RT-PCR), ligase chain reaction, branched DNA signalamplification, transcription-based amplification, amplifiable RNAreporters, Q-beta replication, boomerang DNA amplification, stranddisplacement activation, cycling probe technology, isothermal nucleicacid sequence based amplification (NASBA) and self-sustained sequencereplication assays. The primers used may be selected for their abilityto characterize the tumor; and (C) detection of the specific amplifiedRNA.

This method of detection can be employed in various methods of useincluding the detection of early cancers and premalignant neoplasms andinvasive or advanced cancers, and for the monitoring of patients duringtreatment therapy and for post-operative monitoring, and to developappropriate patient-specific treatment strategies as described herein.

DETAILED DESCRIPTION OF THE INVENTION

The use of RNA detection is preferred in many circumstances over DNAdetection since a greater number of RNA molecules are potentiallyavailable, thus allowing potentially greater sensitivity. Furthermore,since wild-type DNA genetic information is identical in all somaticcells of an individual, discrimination between normal andtumor-associated DNA is dependent upon the presence of a mutation.Detection of RNA, by reflecting activity of the gene, allowsdemonstration of an inappropriately expressing non-mutated gene, as istypically seen in malignancy. Thus, RNA amplification methods allow away to detect gene expression, whether normal or mutated, which isturned on in cancer. The present invention provides a much greaterapplicability and versatility to monitoring cancer than do any methodsbased on DNA analysis. For a DNA method to detect cancers from normals,there must be some mutation or genetic rearrangement present in thecancer, but not in the normal. The present process of using RNA willsimilarly detect the mutant RNA produced from this DNA. However, itfurther allows detection of inappropriately expressing “normal” genes.Thus, compared to methods detecting DNA, methods detecting RNA providegreater versatility and applicability in addition to the expectedgreater sensitivity.

This invention relates to a method of detecting or inferring thepresence of cancerous or precancerous cells, whether from anon-hematologic malignancy (i.e., solid tumor) or from a hematologicmalignancy, in a human or animal by the combination of three stepsapplied to plasma or serum. The first step (Part A) involves theextraction of tumor-derived or associated RNA from blood plasma orserum. The second step (Part B) applies a nucleic acid amplificationassay to the extracted RNA. In this step, the extracted RNA may first bereverse transcribed to cDNA prior to amplification of the cDNA. Thethird step (Part C) allows for the detection of the amplified product.Parts B and C may be performed as to allow either qualitative orquantitative detection of the RNA, depending upon the ultimate clinicalobjective or application, as described herein. Various methods, as,detailed below, may be used in Part A. Similarly, any nucleic acidamplification assay which can be utilized in the detection of smallnumbers of RNA or corresponding cDNA molecules, including but notlimited to the polymerase chain reaction (RT-PCR), branched DNA signalamplification, ligase chain reaction, isothermal nucleic acid sequencebased amplification (NASBA), Q-beta replication, transcription-basedamplification, amplifiable RNA reporters, boomerang DNA amplification,strand displacement activation, cycling probe technology, and otherself-sustained sequence replication assays, as well as variations onthese including methods for nucleic acid enrichment such as by usingrestriction digestion with polymerase chain reaction and the use ofnested primers, may be used in Part B. Similarly, any method capable ofdemonstrating amplified nucleic acid product, including but not limitedto agarose gel electrophoresis, ELISA detection methods,electrochemiluminescence, high performance liquid chromatography, andreverse dot blot methods, may be used in Part C. In this invention, anyof the various. Methods in Part A may be combined with any methodapplicable for Part B, which can then be combined with any applicablemethod in Part C. It is the new application of these methods to thedetection of tumor-derived or associated RNA in plasma or serum, and inparticular to extracellular RNA but also to plasma or serumintracellular RNA, that makes this invention novel. Several methodsapplicable for each of Part A, Part B, and Part C, will be described indetail below as a description of the invention. Again, it is to beemphasized that any method in Part A can be combined with any method inPart B, with any method in Part C to follow. Furthermore, it should beemphasized that while the contribution of extracellular RNA versusintracellular RNA as detected in plasma or serum may be defined, forexample by using filters or high speed centrifugation, it is not arequirement of the invention that such a definition be made.

Either “fresh” blood plasma or serum, or frozen (stored) andsubsequently thawed plasma or serum may be used for purposes of thisinvention. Frozen (stored) plasma or serum should optimally bemaintained at storage conditions of −20 to −70 degrees centigrade untilthawed and used. “Fresh” plasma or serum should be refrigerated ormaintained on ice until used, with RNA extraction being performed assoon as possible.

Blood is drawn by standard methods into a collection tube, preferablysiliconized glass, either without anticoagulant for preparation ofserum, or with EDTA, sodium citrate, heparin, or similar anticoagulantsfor preparation of plasma. The preferred method if preparing plasma orserum for storage, although not an absolute requirement, is that plasmaor serum be first fractionated from whole blood prior to being frozen.This reduces the burden of extraneous intracellular RNA released fromlysis of frozen and thawed cells which might reduce the sensitivity ofthe amplification assay or interfere with the amplification assaythrough release of inhibitors to PCR such as porphyrins and hematin.“Fresh” plasma or serum may be fractionated from whole blood bycentrifugation, using preferably gentle centrifugation at 300-800×g forfive to ten minutes, or fractionated by other standard methods. Highcentrifugation rates capable of fractionating out apoptotic bodiesshould be avoided. Since heparin may interfere with RT-PCR, use ofheparinized blood may require pretreatment with heparinase as described(23), followed by removal of calcium prior to reverse transcription, asdescribed (23). Thus, EDTA is the preferred anticoagulant for bloodspecimens in which PCR amplification is planned.

Part A: Extraction of Extracellular RNA from Plasma or Serum

In Part A, RNA extraction methods previously published for theextraction of mammalian intracellular RNA or viral RNA may be adapted,either as published or with modification, for extraction oftumor-derived or associated RNA from plasma and serum. The volume ofplasma or serum used in part A may be varied dependent upon clinicalintent, but volumes of 100 microliters to one milliliter of plasma orserum are sufficient in part A, with the larger volumes often indicatedin settings of minimal or premalignant disease. For example:

Both extracellular RNA and intracellular RNA may be extracted fromplasma or serum using silica particles, glass beads, or diatoms, as inthe method or adaptations of Boon et al. (24). Application of the methodadapted by Cheung et al. (25) is described:

Size fractionated silica particles are prepared by suspending 60 gramsof silicon dioxide (SiO₂, Sigma Chemical Co., St. Louis, Mo.) in 500milliliters of demineralized sterile double-distilled water. Thesuspension is then settled for 24 hours at room temperature.Four-hundred thirty (430) milliliters of supernatant is removed bysuction and the particles are resuspended in demineralized, steriledouble-distilled water added to equal a volume of 500 milliliters. Afteran additional 5 hours of settlement, 440 milliliters of the supernatantis removed by suction, and 600 microliters of HCl (32% wt/vol) is addedto adjust the suspension to a pH2. The suspension is aliquotted andstored in the dark.

Lysis buffer is prepared by dissolving 120 grams of guinidinethiocyanate (GuSCN, Fluka Chemical, Buchs, Switzerland) into 100milliliters of 0.1 M Tris hydrochloride (Tris-HCl) (pH 6.4), and 22milliliters of 0.2 M EDTA, adjusted to pH 8.0 with NaOH, and 2.6 gramsof Triton X-100 (Packard Instrument Co., Downers Grove, Ill.). Thesolution is then homogenized.

Washing buffer is prepared by dissolving 120 grams of guinidinethiocyanate (GuSCN) into 100 milliliters of 0.1 M Tris-HCl (pH 6.4).

One hundred microliters to two hundred fifty microliters (with greateramounts required in settings of minimal disease) of plasma or serum aremixed with 40 microliters of silica suspension prepared as above, andwith 900 microliters of lysis buffer, prepared as above, using anEppendorf 5432 mixer over 10 minutes at room temperature. The mixture isthen centrifuged at 12,000×g for one minute and the supernatantaspirated and discarded. The silica-RNA pellet is then washed twice with450 microliters of washing buffer, prepared as above. The pellet is thenwashed twice with one milliliter of 70% (vol/vol) ethanol. The pellet isthen given a final wash with one milliliter of acetone and dried on aheat block at 56 degrees centigrade for ten minutes. The pellet isresuspended in 20 to 50 microliters of diethyl procarbonate-treatedwater at 56 degrees centigrade for ten minutes to elute the RNA. Thesample can alternatively be eluted for ten minutes at 56 degreescentigrade with a TE buffer consisting of 10 millimolar Tris-ris-HCl-onemillimolar EDTA (pH 8.0) with an RNase inhibitor (RNAsin, 0.5U/microliter, Promega), with or without Proteinase K (100 ng/ml) asdescribed by Boom et al. (26). Following elution, the sample is thencentrifuged at 12,000×g for three minutes, and the RNA containingsupernatant recovered. The RNA extract is now used in Part B.

As an alternative method, both extracellular RNA and intracellular RNAmay be extracted from plasma or serum in Part A using the AcidGuanidinium Thiocyanate-Phenol-chloroform extraction method described byChomozynski and Sacchi (27) as follows:

The denaturing solution consists of 4 M guanidinium thiocyanate, 25millimolar sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M2-mercaptoethanol. The denaturing solution is prepared as follows: Astock solution is prepared by dissolving 250 grams of guanidiniumthiocyanate (GuSCN, Fluka Chemical) with 293 milliliters ofdemineralized sterile double-distilled water, 17.6 milliliters of 0.75 Msodium citrate, pH 7.0, and 26.4 milliliters of 10% sarcosyl at 65degrees centigrade. The denaturing solution is prepared by adding 0.36milliliters 2-mercaptoethanol/50 milliliters of stock solution.

One hundred microliters to one milliliter of plasma or serum is mixedwith one milliliter of denaturing solution. Sequentially, 0.1 milliliterof 2 M sodium acetate, pH 4.0, 1 milliliter of phenol, and 0.2milliliter of chloroform-isoamyl alcohol (49:1) are added, with mixingafter addition of each reagent. The resultant mixture is shakenvigorously for 10 seconds, cooled on ice for 15 minutes, and thencentrifuged at 10,000×g for 20 minutes at 4 degrees centigrade. Theaqueous phase is then transferred to a clean tube and mixed with 1milliliter of isopropanol. The mixture is then cooled at −20 degreescentigrade for 1-2 hours to precipitate RNA. After centrifugation at10,000×g for 20 minutes the resulting RNA pellet is dissolved in 0.3milliliter of denaturing solution, and then reprecipitated with 1 volumeisopropanol at −20 degrees centigrade for one hour. Following anothercentrifugation at 10,000×g for ten minutes at 4 degrees centigrade, 75%ethanol is added to resuspend the RNA pellet, which is then sedimentedand vacuum dried, and then dissolved in 5-25 microliters of 0.5% SDS at65 degrees centigrade for ten minutes. The RNA extract is now used inPart B.

As the preferred embodiment for Part A, and as an alternative method,extracellular RNA and intracellular RNA may be extracted from plasma orserum in Part A using variations of the acid guanidiniumthiocyanate-phenol-chloroform extraction method. For example, in thepreferred embodiment RNA is extracted from plasma or serum using TRIreagent, a monophase guanidine-thiocyanate-phenol solution, as describedby Chomczynski (28). One hundred microliters to one milliliter of plasmaor serum is processed using one milliliter of TRI Reagent(TM) (TRIReagent, Sigma Trisolv(TM), BioTecx Laboratories, Houston, Tex.,TRIzol(TM), GIBCO BRL/Life Technologies, Gaithersburg, Md., ISOGEN(TM),Nippon Gene, Toyama, Japan, RNA Stat(TM) 60, Tel-test, Friendsword,Tex.) according to manufacturer's directions. Minor adaptations may beapplied as currently practiced within the art. Thus, from one hundredmicroliters to one milliliter of plasma or serum is mixed with onemilliliter of TRI Reagent. Then 0.2 milliliter of chloroform is mixedfor 15 seconds, and the mixture allowed to stand for 3 minutes at roomtemperature. The mixture is then centrifuged at 4 degrees centigrade for15 minutes at 12,000×g. The upper aqueous phase is removed to which 0.5milliliter of isopropanol is mixed, and then left at room temperaturefor five minutes, followed by centrifugation at 4 degrees centigrade forten minutes at 12,000×g. The RNA pellet is then washed with onemilliliter of 75% ethanol by centrifuging at 12,000×g for 5 minutes. Thepellet is air dried and resuspended in 11.2 microliters of RNAse freewater. Contamination by polysaccharides and proteoglycans, which may bepresent in extracellular proteolipid-RNA complexes, may be reduced bymodification of the precipitation step of the TRI Reagent(TM) procedure,as described by Chomczynski and Mackey (29) as follows:

One hundred microliters to one milliliter of plasma or serum is mixedwith TRI Reagent(TM) as per manufacturer's directions, being subjectedto phase separation using either chloroform or bromo-chloropropane (30)and centrifugation at 10,000×g for 15 minutes. The aqueous phase isremoved and then mixed with 0.25 milliliters of isopropanol followedwith 0.25 milliliters of a high-salt precipitation solution (1.2 M NaCland 0.8 M sodium citrate). The mixture is centrifuged at 10,000×g for 5minutes and washed with one milliliter of 75% ethanol. The RNA pellet isthen vacuum dried and then dissolved in 5-25 microliters of 0.5% SDS at65 degrees centigrade for ten minutes. The RNA extract is now used inPart B.

Alternative methods may be used to extract RNA from plasma or serum inPart A, including but not limited to centrifugation through a cesiumchloride gradient, including the method as described by Chirgwin et al.(31), and co-precipitation of extracellular RNA from plasma or serumwith gelatin, such as by adaptations of the method of Fournie et al.(32) to RNA extraction.

Circulating extracellular deoxyribonucleic acid (DNA), includingtumor-derived or associated extracellular DNA, is also present in plasmaand serum (33). Since this DNA will additionally be extracted to varyingdegrees during the RNA extraction methods described above, it may bedesirable or necessary (depending upon clinical objectives) to furtherpurify the RNA extract and remove trace DNA prior to proceeding to PartB. This may be accomplished using DNase, for example by the method asdescribed by Rashtchian (34), as follows:

For one microgram of RNA, in a 0.5 milliliter centrifuge tube placed onice, add one microliter of 10×DNase I reaction buffer (200 micromolarTris-HCl (pH 8.4), 500 micromolar KCl, 25 micromolar MgCl₂, onemicromolar per milliliter BSA). Add to this one unit DNase I (GIBCO/BRLcatalog #18068-015). Then bring the volume to ten microliter withDEPC-treated distilled water, and follow by incubating at roomtemperature for 15 minutes. The DNase I is then inactivated by theaddition of 20 millimolar EDTA to the mixture, and heating for 10minutes at 65 degrees centigrade. The treated RNA may now go directly toPart B.

Alternatively, primers in Part B may be constructed which favoramplification of the RNA products, but not of contaminating DNA, such asby using primers which span the splice junctions in RNA, or primerswhich span an intron. Alternative methods of amplifying RNA but not thecontaminating DNA include the methods as described by Moore et al. (35),and methods as described by Buchman et al. (36), which employs adU-containing oligonucleotide as an adaptor primer.

Part B: Nucleic Acid Amplification

In Part B, RNA which has been extracted from plasma or serum during PartA, or its corresponding cDNA, is amplified using any nucleic acidamplification assay utilized for detection of low numbers of RNAmolecules. Applicable assays include but are not limited to reversetranscriptase polymerase chain reaction (RT-PCR), ligase chain reaction(37), branched DNA signal amplification (38), amplifiable RNA reporters,Q-beta replication, transcription-based amplification, boomerang DNAamplification, strand displacement activation, cycling probe technology,isothermal nucleic acid sequence based amplification (NASBA) (39), andother self-sustained sequence replication assays. It is not necessary tomodify these assays from their published methods for Part B. Thereferenced publications are incorporated herein by reference in theirentirety for their descriptions for performing the various assaysidentified therein. It is the application of these nucleic acidamplification assays to the detection of tumor-derived or associatedextracellular RNA in plasma or serum that makes their use novel. Thepreferred embodiment for Part B uses the reverse transcriptasepolymerase chain reaction (RT-PCR).

Primers used in the amplification assay should be based on the specifictumor-derived or associated RNA of interest which characterizes thetumor. Tumor-derived or associated RNA includes but is not limited to:

-   -   mRNA related to mutated oncogenes or mutated DNA, a partial list        of which includes H-ras, K-ras, N-ras, c-myc, her-2-neu,        bcr-abl, fms, src, fos, sis, jun, erb-B-1, VHL, PML/RAR,        AML1-ETO, EWS/FLI-1, EWS/ERG.    -   mRNA related to tumor suppressor genes, a partial list of which        includes p53, RB, MCC, APC, DCC, NF1, WT.    -   mRNA related to tumor-associated protein which is found elevated        in certain cancers, a partial list of which includes alpha-feto        protein (AFP), carcinoembryonic antigen (CEA), TAG-72, CA 19-9,        CA-125, prostate specific antigen (PSA), CD44, and hcg (human        chorionic gonadotropin).    -   mRNA related to tumor-derived protein not normally found        circulating in blood, a partial list of which includes        tyrosinase mRNA, keratin 19 mRNA.    -   mRNA related to tumor-specific antigens, such as in MAGE 1, MAGE        2, MAGE 3, MAGE 4, GP-100, and MAGE 6, MUC 18, P97.    -   mRNA or messenger-like RNA associated with ribonucleoproteins        and RNA within ribonucleoproteins, a partial list of which        includes telomerase RNA, and RNA associated with heterogenous        nuclear ribonucleoprotein Al (hn RNP-A1) and A2/B1 (hn        RNP-A2/B1) complexes, and heterogenous nuclear ribonucleoprotein        K (hn RNP-K), such as c-myc oncogene RNA, in addition to those        RNA previously described above when associated with        ribonucleoprotein.

For example, oligonucleotide primer sequences for the bcr-abl transcriptmay be as follows (40):

Primer 1 at the M-bar location: (5′-TGGAGCTGCAGATGCTGACCAACTCG-3′). SEQID NO: 1 Primer 2 at the exon II abl location:(5′-ATCTCCACTGGCCACAAAATCATACA-3′). SEQ ID NO: 2 Primer 3 at the M-barlocation: (5′-GAAGTGTTTCAGAAGCTTCTCC-3′). SEQ ID NO: 3 Primer 4 at theexon II abl location: (5′-TGATTATAGCCTAAGACCCGGA-3′). SEQ ID NO: 4

The nested RT-PCR assay yields a 305 or a 234 base pair product,depending upon bcr exon 3 expression.

As another example, nested primers for human tyrosinase cDNAamplification can be as follows (41):

Primer 1 (outer, sense) - (5′-TTGGCAGATTGTCTGTAGCC-3′) SEQ ID NO: 5Primer 2 (outer, anti-sense) - (5′-AGGCATTGTCCATGCTGCTT-3′) SEQ ID NO: 6Primer 3 (nested, sense) - (5′-GTCTTTATGCAATGGAACGC-3′) SEQ ID NO: 7Primer 4 (nested, anti-sense) - (5′-GCTATCCCAGTAAGTGGACT-3′) SEQ ID NO:8

The outer primers result in a PCR amplification product of 284 basepairs, and the nested primers result in a fragment of 207 base pairs.

The preferred oligonucleotide primer sequences for specifictumor-related or tumor-associated mRNA are previously published, withreferenced publications incorporated herein by reference in theirentirety.

Some, but not all, amplification assays require reverse transcription ofRNA to cDNA. As noted, the method of reverse transcription andamplification may be performed by previously published or recommendedprocedures, which referenced publications are incorporated herein byreference in their entirety, and modification is not required by theinvention beyond steps as described in Part A. Various reversetranscriptases may be used, including, but not limited to, MMLV RT,RNase H mutants of MMLV RT such as Superscript and Superscript II (LifeTechnologies, GIBCO BRL, Gaithersburg, Md.), AMV RT, and thermostablereverse transcriptase from Thermus Thermophilus. For example, onemethod, but not the only method, which may be used to convert RNAextracted from plasma or serum in Part A to cDNA is the protocol adaptedfrom the Superscript II Preamplification system (Life Technologies,GIBCO BRL, Gaithersburg, Md.; catalog no. 18089-011), as described byRashtchian (34), adapted as follows:

1-5 micrograms of RNA extracted from plasma or serum in Part A in 13microliters of DEPC-treated water is added to a clean microcentrifugetube. Then one microliter of either oligo (dT) (0.5milligram/milliliter) or random hexamer solution (50 ng/microliter) isadded and mixed gently. The mixture is then heated to 70 degreescentigrade for 10 minutes and then incubated on ice for one minute.Then, it is centrifuged briefly followed by the addition of 2microliters of 10×synthesis buffer (200 mM Tris-HCl, pH 8.4, 500 mM KCl,25 mm magnesium chloride, one milligram/milliliter of BSA), onemicroliter of 10 mM each of dNTP mix, 2 microliters of 0.1 M DTT, onemicroliter of SuperScript II RT (200 U/microliter) (Life Technologies,GIBCO BRL, Gaithersburg, Md.). After gentle mixing, the reaction iscollected by brief centrifugation, and incubated at room temperature forten minutes. The tube is then transferred to a 42 degrees centigradewater bath or heat block and incubated for 50 minutes. The reaction isthen terminated by incubating the tube at 70 degrees centigrade for 15minutes, and then placing it on ice. The reaction is collected by briefcentrifugation, and one microliter of RNase H (2 units) is addedfollowed by incubation at 37 degrees centigrade for 20 minutes beforeproceeding to nucleic acid amplification.

Nucleic acid amplification then proceeds as follows:

To the cDNA mixture add the following: 8 microliters of 10×synthesisbuffer (200 mM Tris-HCl, pH 8.4, 500 mM KCl, 25 mM magnesium chloride, 1mg/ml of BSA), 68 microliters sterile double-distilled water, onemicroliter amplification primer 1 (10 micromolar), one microliteramplification primer 2 (10 micromolar), one microliter Taq DNApolymerase (2-5 U/microliter). Mix gently and overlay the reactionmixture with mineral oil. The mixture is heated to 94 degrees centigradefor 5 minutes to denature remaining RNA/cDNA hybrids. PCR amplificationis then performed in an automated thermal-cycler for 15-50 cycles, at 94degrees centigrade for one minute, 55 degrees centigrade for 30 to 90seconds, and 72 degrees centigrade for 2 minutes. The amplified PCRproduct is then detected in Part C.

Furthermore, if the primers contain appropriate restriction sites,restriction digestion may be performed on the amplified product to allowfurther discrimination between mutant and wild-type sequences.

Cycling parameters and magnesium concentration may vary depending uponthe specific case. For example, an alternative method using nestedprimers useful for detection of human tyrosinase mRNA in Part B is themethod described by Smith et al. (4), as follows:

Primer sequences are as described above for human tyrosinase. Tenmicroliters of RNA extracted in Part A from plasma or serum are treatedfor reverse transcription by heating at 90 degrees centigrade for 4minutes, cooling rapidly, and diluting to 20 microliters with a mixtureconsisting of 1×PCR buffer (10 mmol/liter Tris-HCl, pH 8.4, 50mmol/liter KCl, 100 microgram/milliter gelatin), 8 mmol/liter magnesiumchloride, 1 mmol/liter each DATP, dCTP, dGTP, and dTTP, 25 pmoltyrosinase primer 2 (as previously described), 20 units of ‘RNA guard’(Pharmacia), and 4 units of murine moloney leukemia virus reversetranscriptase (Pharmacia). The total mixture is then incubated at 37degrees centigrade for one hour, half the sample removed, and diluted to50 microliters containing 1×PCR buffer, 200 micromol/liter each of dATP,dCTP, dGTP, and dTTP, 1.6 mmol/liter magnesium chloride, 150 pmol primer1 and primer 2, 0.1% Triton X-100, and 1 unit Taq DNA polymerase(Promega). The mixture is overlaid with oil, and heated at 95 degreescentigrade for 5 minutes, followed by 30 cycles of PCR in a thermalcycler at 95 degrees centigrade for 65 seconds, 55 degrees centigradefor 65 seconds, and 72 degrees centigrade for 50 seconds. The productsare then reamplified with nested primer 3 and nested primer 4 using 5microliters in a 1:100 dilution. These were amplified in a 25 microliterreaction volume for an additional 30 cycles. This final amplified PCRproduct is now detected in Part C, either by being electrophoresed on anagarose gel, or by other method.

The preferred embodiments for Part B amplification of specifictumor-related or tumor-associated RNA, including specific primers,method of reverse transciption, and method of RT-PCR, are described bythe following referenced publications which are incorporated herein byreference in their entirety for their description for performing thevarious assays identified therein.

For Part B amplification of tyrosinase mRNA, a mRNA associated withmalignant melanoma, the preferred method is that of Brossart et al.(41).

For Part B amplification of Keratin 19 mRNA, a mRNA associated withbreast cancer and other epithelial malignancies, the preferred method isthat of Datta et al. (5).

For Part B amplification of prostate-specific antigen (PSA) mRNA, a mRNAassociated with prostate cancer, the preferred method is that of Katz,et al. (72).

For Part B amplification of alpha-fetoprotein (AFP) mRNA, a mRNAassociated with hepatocellular carcinoma, testicular cancer, and othercancers, the preferred method is that of Komeda et al. (54).

For Part B amplification of BCR/abl mRNA, a mRNA associated with chronicmyeloid leukemia (CML), the preferred method is that of Stock et al.(57), or alternatively, by the method of Edmonds et al. (40).

For Part B amplification of carcinoembryonic antigen (CEA) mRNA, a mRNAassociated with gastrointestinal cancers and breast cancer, thepreferred method is that of Gerhard et al. (58).

For Part B amplification of P97 mRNA, a mRNA associated with malignantmelanoma, the preferred method is that of Hoon et al. (59).

For Part B amplification of MUC 18 mRNA, a mRNA associated withmalignant melanoma, the preferred method is that of Hoon et al. (59).

For Part B amplification of PML/RAR −α mRNA, a mRNA associated withacute promyelocytic leukemia, the preferred method is that of Miller etal. (60).

For Part B amplification of CD44 mRNA, a mRNA associated with lungcancer, the preferred method is that of Penno et al. (61).

For Part B amplification of EWS/FLI-1 mRNA, a mRNA associated withEwing's sarcoma and other Ewing's tumors, the preferred method is thatof Pfleiderer et al. (62).

For Part B amplification of EWS/ERG mRNA, a mRNA associated with Ewing'ssarcoma and other Ewing's tumors, the preferred method is that ofPfleiderer et al. (62).

For Part B amplification of AML1/ETO mRNA, a mRNA associated with acutemyelogenous leukemia, the preferred method is that of Maruyama et al.(63).

For Part B amplification of MAGE mRNA, including mRNA of MAGE-1, MAGE-2,MAGE-3, and MAGE-4, which are associated with bladder cancer, ovariancancer, melanoma, lung cancer, head and neck cancer, and others, thepreferred method is that of Patard et al. (64).

For Part B amplification of beta-human chorionic gonadotropin mRNA, amRNA associated with malignant melanoma, germ cell tumors, and othercancers, the preferred method is that of Doi et al. (65).

For Part B amplification of human Telomerase-associated RNA, thepreferred method is by application of the TRAP PCR method as describedby Kim et al (69). Alternatively, other amplification methods may beused as described herein where primer selection is designed based uponthe human Telomerase template sequence as described by Peng et al (76).

Alternative methods of nucleic acid amplification which may be used inPart B include other variations of RT-PCR, including quantitativeRT-PCR, for example as adapted to the method described by Wang et al.(43) or by Karet et al. (44).

An alternative method of nucleic acid amplification which may be used inPart B is ligase chain reaction (66). Extracellular RNA extracted fromplasma or serum in Part A must be reverse transcribed to cDNA.Oligonucleotide primers are selected which lie directly upon the cDNAsite of interest. If a mutation site is present, oligonucleotides whichare complementary to the site are made to contain the mutation only attheir 3-prime end, excluding hybridization of non-mutated, wild-typeDNA. Restriction sites can also be utilized to discriminate betweenmutant and wild-type sequences if necessary.

An alternative method of either qualitative or quantitativeamplification of nucleic acid which may be used in Part B is branchedDNA signal amplification, for example as adapted to the method describedby Urdea et al. (38), with modification from the reference as follows:plasma or serum should only be centrifuged at lower speeds, aspreviously outlined. Extracellular RNA is then extracted from plasma orserum as described in Part A, and then added directly to microwells. Themethod for detection of tumor-related or tumor-associated RNA thenproceeds as referenced (38), with target probes specific for thetumor-related or tumor-associated RNA or cDNA of interest, and withchemiluminescent light emission proportional to the amount oftumor-associated RNA in the plasma or serum specimen. The specifics ofthe referenced method are described further bu Urdea et al (71) withthis reference incorporated herein in its entirety.

An alternative method of either qualitative or quantitativeamplification of nucleic acid which may be used in Part B is isothermalnucleic acid sequence based amplification (NASBA), for example asadapted to the method described by Kievits et al. (39), or by Vandammeet al. (45). The method of Sooknanan et al. (67) may be used for thedetection and quantification of BCR/ABL mRNA.

Alternative methods of either qualitative or quantitative amplificationof nucleic acids which may be used in Part B include, but are notlimited to, Q-beta replication, other self-sustained sequencereplication assays, transcription-based amplification assays, andamplifiable RNA reporters, boomerang DNA amplification, stranddisplacement activation, and cycling probe technology.

The amplified product from Part B is next detected in Part C. Dependingupon the detection method used in Part C, primers may need to bebiotinylated or otherwise modified in Part B.

Part C: Detection of Amplified Product

There are numerous methods to detect amplified nucleic acid product, anyof which may be used in Part C to detect the amplified product from PartB. The referenced publications, including those pertaining to detectionof specific tumor-related or associated RNA or its corresponding cDNA aspreviously cited, and those pertaining to RNA or its corresponding cDNAdetection as follows, are incorporated herein by reference in theirentirety for the descriptions for performing the various assaysidentified therein.

In the preferred method, amplified product is detected in Part C usinggel electrophoresis. In the preferred embodiment, 25 microliters ofamplified (or post-amplification digested) product is electrophoresedthrough a 3% agarose gel in 1×TBE at 75 VDC. Electrophoresis is carriedout for one to two hours before staining with ethidium bromide. As analternative to ethidium bromide, the amplified product can betransferred from the gel to a membrane by blotting techniques to bedetected with a labeled probe (46).

An alternative method which may be used in Part C to detect theamplified product from Part B is ELISA detection. Depending upon theELISA detection method used, it may be necessary to biotinylate orotherwise modify the primers used in part B.

For example, one ELISA detection method which may be used in Part C isthe method described by Landgraf et al. (47), as follows:

Primers are modified with biotinylamidocaproat-N-hydroxysuccinimidester(Sigma) and fluoroescein isothiocyanate (FITC) (Sigma) by the method ofLandgraf et al. (48). Following invention Part B, the ELISA is carriedout in microtiter plates coated with 1 microgram/milliliteraffinity-purified avidin (13 U/mg, Sigma). One microliter of the finalamplification product (or post-digestion product) is diluted with 50microliters of PBS-Tween, and then incubated at room temperature for 30minutes in the microtiter plate well. Non-incorporated primers areremoved by washing with PBS-Tween. The plates are then incubated at roomtemperature for 30 minutes after adding 50 microliters per well ofanti-FITC antibody-HRPO conjugate (Dakopatts) which Is at a 1:500dilution with PBS-Tween. Following this, 80 microliters of an ELISAsolution made from one milligram 3, 3′, 5, 5′-tetramethylbenzidin(Sigma) dissolved in one milliliter dimethyl sulfoxide, and diluted 1:10with 50 millimol Na-acetate: citric acid, pH 4.9, with 3 microliter of30% (vol/vol) H₂O₂ added, is added to each well. After 2-5 minutes, thereaction is stopped by adding 80 microliter of 2 M H₂O₄. The opticaldensity is then read at 450 nm.

Alternative methods of ELISA detection which may be used in Part Cinclude, but are not limited to, immunological detection methods usingmonoclonal antibody specific for RNA/DNA hybrids, such as by adaptingmethods described by Coutlee et al. (49), or by Bobo et al. (50), whichpublications are also incorporated herein by reference in their entiretyfor their description of the detection methods identified therein.

Alternative methods of ELISA detection which may be used in Part Cinclude, but are not limited to, commercial detection systems such asthe SHARP signal system (Digene Diagnostics, Inc.), and the DNA enzymeimmunoassay (DEIA), (GEN-ETI-K DEIA, Sorin Biomedica).

Alternative methods by which amplified product from Part B may bedetected in Part C include but are not limited to all methods ofelectrochemiluminescence detection, such as by adapting the methoddescribed by Blackburn et al. (51), or by DiCesare et al. (52), and allmethods utilizing reverse dot blot detection technology (53), and allmethods utilizing high-performance liquid chromatography.

Therapeutic Applications

The extraction of extracellular tumor-associated or derived RNA fromplasma or serum, and the amplification of that RNA or its correspondingcDNA to detectable levels, permits further analysis or othermanipulation of that RNA, or the corresponding cDNA, from which furtherclinical utility is realized. In this optional step of the invention,amplified extracellular RNA or the corresponding cDNA is analyzed todefine the characteristics or composition of the tumor from which theRNA originates. Any of several methods may be used, dependent upon thedesired information, including. Nucleic acid sequencing, spectroscopyincluding proton NMR spectroscopy, biochemical analysis, and immunologicanalysis. In the preferred embodiment, amplified cDNA is isolated—forexample by excising DNA bands from an agarose gel—reamplified, clonedinto a plasmid vector, for example the PGEM-T vector plasmid (Promega)and sequenced using a commercial kit such as Sequenase 2.0 (USB).Analysis to define the characteristics or composition of thetumor-associated RNA in plasma or serum, and thus the tumor of origin,affords a wide array of clinical utility, including the description,characterization, or classification of the tumor, whether known oroccult, such as by tissue of origin, by type (such as premalignant ormalignant), phenotype, and genotype, and by description orcharacterization of tumor behavior, physiology and biochemistry, as togain understanding of tumor invasiveness, propensity to metastasize, andsensitivity or resistance to various therapies, thereby allowing theprediction of response to either ongoing or planned therapy and,further, allowing evaluation of prognosis. Comparison of thecharacteristics of extracellular RNA to previous biopsy or surgicalspecimens permits further evaluation of tumor heterogeneity orsimilarity in comparison to that specimen, and thus evaluation of tumorrecurrence.

Following extraction of extracellular tumor-derived or tumor-associatedRNA from plasma or serum and amplification of the corresponding cDNA,ribonucleic acid (RNA) may be transcribed or manufactured back from theamplified DNA as a further option. Transcription of RNA may be performedby employing a primer with an RNA polymerase promoter region joined tothe standard primer sequence of the cDNA in an amplification reaction.RNA is then transcribed from the attached promoter region. In thepreferred embodiment, amplified cDNA is cloned into an expressionvector, and RNA is transcribed. Furthermore, as an optional preferredembodiment, the RNA is used in an in vitro translation reaction tomanufacture tumor-associated or tumor-specific protein or associatedpeptides or oligopeptides, according to methods currently known in theart (73-76). Note, these cited references, and those to follow, areincorporated herein by reference in their entirety for their descriptionfor performing the various assays identified therein.

Extraction of tumor-derived or tumor-associated extracellular RNA, itsamplification, characterization, and translation to tumor-associated ortumor-specific protein, provides significant utility, both in theassignment of therapy and in the development of tumor-specifictherapies. Sequencing of RNA or cDNA allows assignment or development ofantisense compounds, including synthetic oligonucleotides and otherantisense constructs appropriately specific to the DNA, such as byconstruction of an expression plasmid such as by adapting the method ofAoki et al. (68) which is incorporated by reference in its entirety, orby other construction and use as referenced (77-81). Thus, applicationof the invention in this manner would entail the extraction oftumor-associated RNA from plasma or serum, followed by an optional stepof reverse transcribing to cDNA, followed by amplification of the RNA orcDNA. The amplified product can then be sequenced to define the nucleicacid sequence of the tumor-associated RNA or cDNA. An antisenseoligonucleotide is then constructed in such a manner as referenced abovespecific to the defined sequence, or alternatively, an alreadymanufactured antisense compound is determined to be applicable, or maybe manufactured when the sequence is known based upon knowledge of theprimer sequence. Similarly, defining tumor characteristics by analysisof extracellular RNA allows assignment of specific monoclonal antibodyor vaccine therapies appropriately specific to the tumor. Production ofcorresponding immunologic protein can be used in the development oftumor-specific monoclonal antibodies. Thus, application of the inventionin this manner would entail the extraction of tumor-associated RNA fromplasma or serum, followed by amplification to obtain a tumor-associatedamplified product. The amplified product is translated, or transcribedand translated, into a protein or associated peptides or oligopeptidesas previously described, thus providing a tumor-associated antigen. Thetumor-associated antigen thus enables production of a monoclonalantibody directed against the antigen by use of hybridoma technology orother methods as currently practiced by the art (82). Said monoclonalantibody may further be conjugated with a toxin or other therapeuticagent (83), or with a radionucleotide (84) to provide furthertherapeutic or diagnostic use directed against the tumor. Similarly,translated protein or associated peptides or oligopeptides can be usedin tumor-specific vaccine development. Furthermore, the extracellularRNA and complimentary DNA permit a means of defining or allowing theconstruction of a DNA construct which may be used in vaccine therapy.Specifically, the invention is applied to either define or obtaintumor-associated protein or peptides, RNA, or cDNA, by methods aspreviously described, and from which a tumor-directed vaccine may bedeveloped or constructed. The methods by which the vaccine is furtherdeveloped or constructed vary, but are known to the art (85-90), and arereferenced herein in their entirety.

Of particular value, the invention allows the development andapplication of these tumor-specific therapies even when onlypremalignant tumors, early cancers, or occult cancers are present. Thus,the invention allows therapeutic intervention when tumor burden is low,immunologic function is relatively intact, and the patient is notcompromised, all increasing the potential for cure.

Hypothetical Examples of the Invention

In the following examples, illustrative hypothetical clinical cases arepresented to demonstrate the potential clinical use of the invent ion.

Case 1

A 26 year old asymptomatic hypothetical man presents for evaluationafter learning his 37 year old brother was recently diagnosed with coloncancer. Peripheral blood is drawn in order to use the invention toevaluate for the presence of extracellular CEA mRNA in the patient'splasma. Plasma extracellular RNA is extracted during invention Part A bythe Acid Guanidinium thiocyanate-Phenol-chloroform extraction method aspreviously described, followed by qualitative RT-PCR amplification inInvention Part B using CEA mRNA primers as previously described. Theamplification assay as previously described (58) is performed ininvention Part B. The final amplified product is detected by gelelectrophoresis on a 3% agarose gel in invention Part C. Results arepositive in this patient indicating the presence of CEA mRNA in theblood plasma.

CEA has been associated with colon cancer. While colon cancer is highlycurable if diagnosed at an early stage, it is fatal when diagnosed atadvanced metastatic stages. The positive results of the invention forthis patient, in the setting of a strongly positive family history forcolon cancer, are suggestive of either premalignant or malignant coloncancer. It is recommended that the patient undergo colonoscopy, and ifno lesion is found, receive surveillance more frequently than wouldnormally be given.

This hypothetical case illustrates how the invention can be used toscreen high risk patients for cancer, detect either premalignant ormalignant conditions prior to the metastatic state, and play a role inclinical management. While CEA mRNA is associated with other cancers,such as liver cancer, the addition of a multiplex panel approach usingthe invention to detect multiple different tumor-associatedextracellular RNA, including for example K-ras, P53, DCC, and APC RNA,enables clarification as to whether the CEA mRNA is likely associatedwith a colon tumor, and further, whether the findings are consistentwith a premalignant or a malignant tumor.

Case 2

A 33 year old hypothetical woman sees her local dermatologist afternoting a “bleeding mole” on her back. Local excision diagnoses amalignant melanoma of 0.3 millimeter depth. Wide surgical re-excision isperformed, and the patient is told she is likely cured and no furthertherapy is needed. Three months following her surgery the patient seeksa second opinion regarding the need for further therapy. Peripheralblood is drawn to evaluate her plasma for the presence of extracellulartyrosinase messenger RNA by the invention. Plasma extracellular RNA isextracted in invention Part A using the preferred TRI-Reagent method aspreviously described, followed by RT-PCR using nested primers fortyrosinase cDNA in invention Part B as previously described, with ELISAdetection in invention Part C. Invention results detect the presence oftyrosinase mRNA in the patient's plasma. Tyrosinase is common to bothnormal melanocytes and malignant melanoma. However, tyrosinase mRNA doesnot normally circulate in blood, and its presence in plasma indicateslatent malignant melanoma. Consequently, the patient is started onadjuvant therapy with interferon-alpha. Plasma extracellular tyrosinaseRNA levels are subsequently serially followed in a quantitative fashionusing the invention. Blood is drawn from the patient every two months,and plasma extracellular RNA is extracted in invention Part A using thesilica extraction method as previously described. Quantitative RT-PCRamplification for tyrosinase mRNA is then performed in invention Part Busing biotinylated primer using electrochemiluminescence based detectionin invention Part C. Invention data demonstrates a serial rise in thepatient's plasma extracellular tyrosinase mRNA levels. Consequent tothis data, the interferon is stopped, and the patient is enrolled intoan experimental adjuvant therapy protocol.

This hypothetical case illustrates several uses of the invention,including the detection of latent cancer, predicting prognosis andcancer recurrence following surgical excision, determining the need foradditional therapy, evaluating the benefit of therapy and the need tochange therapies, and evaluating prognosis of patients on therapy.

Case 3

A 76 year old hypothetical man is noted to have a pancreatic mass on CTscan imaging. His chest x-ray and colonoscopy are normal. The patientrefuses to consider surgery because of the significant surgical risks.He elects to receive patient-specific therapy made possible by use ofthe invention. Since K-ras mutations are present in 80-90% of pancreaticcancers, peripheral blood is drawn to evaluate for and characterizeextracellular mutant K-ras RNA circulating in plasma using theinvention. Plasma extracellular RNA is extracted in invention Part Ausing the TRI reagent extraction method as previously described,followed by RT-PCR in invention Part B, with high performance liquidchromatography detection in Part C. Mutant K-ras amplification productsare then separated following chromatography and the K-ras mutation issequenced using standard techniques as previously described. Detectionof mutant K-ras mRNA in the plasma confirms the likelihood of thepancreatic mass being a pancreatic cancer. Based upon the mutationsequence, a patient-specific therapy (i.e., specific to the patient'sown cancer) is developed, in this case a ras vaccine specific to themutant oncogene in this patient's pancreatic cancer. Alternatively,mutant K-ras specific protein, generated as previously described, may beused to develop a tumor-specific monoclonal antibody.

In this hypothetical case, the invention is used not only to helpconfirm a suspected diagnosis of pancreatic cancer, but to develop apatient-specific therapy. Patient-specific therapies—i.e., therapiesspecifically designed for a given patient's cancer, or a given type ofcancer, are possible when specific characteristics of the tumor arerecognized. Since the invention results in amplification of pure tumorproduct, it becomes possible to characterize the tumor, in this caseusing sequence analysis and/or transcription and translation. Thetechnological leap that the invention enables is that it allows tumorsto be characterized without the need for biopsy or surgery. Thus, itbecomes possible to treat tumors even before they become clinicallyevident, i.e., treating at latent stages, pre-recurrence stages, or evenpre-malignant stages. Early treatment of cancer before metastatic cellsenter the bloodstream increases the likelihood of cure.

Case 4

A 36 year old hypothetical woman who has three small children at homewas diagnosed with breast cancer two years ago. She had been treatedwith surgery followed by six months of chemotherapy. In addition, herblood serum has been serially evaluated for extracellular keratin 19mRNA using the invention in which serum extracellular kerain 19 mRNA isextracted in invention Part A using the silica extraction method,followed by RT-PCR amplification in invention Part B with ELISAdetection in invention Part C. Keratin 19 mRNA encodes for anintermediate filament protein not normally found in blood which canserve as a marker for breast cancer. While previous results for thispatient had been negative, her blood serum is now testing positive forextracellular keratin 19 mRNA by the invention, suggesting an impendingcancer recurrence. A multiplex panel for serum extracellular myc, ras,P53, EGFr, and Her-2-neu RNA is performed using the invention. This dataconfirms that tumor characteristics are identical to those of theoriginal breast cancer primary, confirming a recurrence rather than anew primary. Consequently, serum extracellular keratin 19 mRNA ismeasured in a quantitative fashion using a branched DNA signalamplification assay in invention Part B, with measurements performed 2months and 4 months later. Quantitative measurements indicate increasinglevels of keratin 19 mRNA, and allow extrapolation to predict thatclinical recurrence will be noted in approximately 2 years. Thisinformation allows both the physician and the patient to plan futuretherapeutic options in the context of the patient's current social andfamily situation.

This hypothetical case illustrates the use of the invention to monitorpatients following therapy for recurrence of their cancer, to determinecharacteristics of their tumor, and to predict prognosis. Breast cancerpatients have a high incidence of second primaries, but the inventionpermits delineation of primary versus recurrent cancer by using amultiplex panel approach to evaluate tumor characteristics. Furthermore,since quantitative analysis in invention Part B allows clarification ofprognosis, the patient is in a better position to plan therapy withinthe context of her social/family situation. Lastly, since the inventionallows detection of tumor-derived extracellular RNA, and does not dependupon the presence of circulating cancer cells, recurrence can bedetected at a very early stage (in this hypothetical case—2 years beforeclinical detection), which increases the likelihood of effectivetherapy.

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1. A method for detecting inappropriate expression or over-expression ofa non-mutated gene in a human or animal, the method comprising the stepsof: a) extracting total RNA from plasma or serum from a human or animal,wherein a fraction of said extracted RNA comprises a RNA species from anon-mutated gene; b) amplifying to produce an amplified fragment orsignal amplifying to produce an amplified signal of said fraction of theextracted RNA or cDNA produced therefrom, wherein amplification isperformed either qualitatively or quantitatively using primers or probesspecific for a portion of the nucleic acid sequence of said RNA or cDNAof said non-mutated genes; c) detecting the amplified fragment oramplified signal produced from the RNA or cDNA thereof, qualitatively orquantitatively, whereby qualitative or quantitative detection of saidfragment or signal indicates inappropriate expression or over-expressionof said non-mutated gene.
 2. The method of claim 1, whereininappropriate expression or over-expression of the non-mutated geneindicates that the human or animal has a disease.
 3. A method fordiagnosing, detecting or monitoring a disease in a human or animal, themethod comprising the step of detecting inappropriate or over-expressionof a non-mutated mRNA according to the method of claim
 1. 4. The methodof claim 3, wherein the disease is a cancer or a premalignant condition.5. A method for screening a human or animal for a disease, the methodcomprising the step of detecting inappropriate or over-expression of anon-mutated mRNA according to the method of claim
 1. 6. The method ofclaim 1, wherein amplified RNA or cDNA is detected by: a) anelectrophoresis-based detection method; b) ELISA detection methods usingbiotinylated or other modified primers; c) immunological detectionmethods using monoclonal antibodies; d) detection methods using alabeled fluorescent or chromagenic probe; e) Southern blot analysis; f)electrochemiluminescence; g) reverse dot blot detection; or h)high-performance liquid chromatography.
 7. The method of claim 1,wherein RNA or cDNA produced therefrom is amplified using a method thatis: a) reverse transcriptase polymerase chain reaction; b) ligase chainreaction; o) branched DNA signal amplification; d) amplifiable RNAreporters; e) Q-beta replication; f) transcription-based amplification;g) isothermal nucleic acid sequence replication assay; h) boomerang DNAamplification; i) strand displacement activation; j) cycling probetechnology; or k) a combination or variation thereof that amplifies theRNA or cDNA.
 8. The method of claim 1, wherein the amplified fragmentproduced from mRNA or cDNA produced therefrom in step b) is furthercharacterized by: a) nucleic acid sequencing; b) spectroscopy; c)immunological analysis; d) production of the corresponding RNA; or e)production of the corresponding protein.
 9. The method of claim 1,wherein the extracted RNA is mammalian RNA.
 10. A method for detecting atumor-associated or tumor-derived RNA species in a plurality of RNAisolated from plasma or serum from a human or animal, wherein thetumor-associated or tumor-derived RNA is non-mutated RNA, the methodcomprising the steps of: a) extracting mammalian total RNA from plasmaor serum, wherein a portion of the extracted RNA comprises atumor-associated or tumor-derived mRNA species of a non-mutated gene; b)amplifying or signal amplifying the non-mutated mRNA or a cDNA producedtherefrom to produce an amplified fragment or amplified signal, whereinamplification is performed qualitatively or quantitatively using primersor probes specific for the tumor-derived or tumor-associated,non-mutated mRNA or cDNA; and c) detecting quantitatively orqualitatively the amplified fragment or amplified signal produced fromthe tumor-derived or tumor-associated, non-mutated mRNA or cDNA.
 11. Themethod of claim 10, wherein RNA or cDNA produced therefrom is amplifiedusing a method that is: a) reverse transcriptase polymerase chainreaction; b) ligase chain reaction; c) branched DNA signalamplification; d) amplifiable RNA reporters: e) Q-beta replication; f)transcription-based amplification; g) isothermal nucleic acid sequencereplication assay; h) boomerang DNA amplification; i) stranddisplacement activation; j) cycling probe technology; or k) acombination or variation thereof that amplifies the RNA or cDNA.
 12. Themethod of claim 10, wherein the amplified fragment is detected by amethod that is: a) an electrophoresis-based detection method b) ELISAdetection methods using biotinylated or other modified primers; c)immunological detection methods using monoclonal antibodies; d)detection methods using a labeled fluorescent or chromagenic probe; e)Southern blot analysis; f) electrochemiluminescence; g) reverse dot blotdetection; or h) high-performance liquid chromatography.
 13. A methodfor detecting, diagnosing or monitoring a malignant or premalignantdisease in a human or animal, the method comprising the step ofdetecting a tumor-derived or tumor-associated, non-mutated mRNA fromplasma or serum obtained from a human or animal using the method ofclaim
 10. 14. A method for screening a human or animal for a malignancyor premalignancy associated with a tumor-associated or tumor-derivedRNA, the method comprising the step of detecting a tumor-derived ortumor-associated, non-mutated mRNA isolated from plasma or serumobtained from a human or animal using the method of claim
 10. 15. Themethod of claim 10, wherein the amplified fragment produced from mRNA orcDNA produced therefrom in step b) is further characterized by: a)nucleic acid sequencing; b) spectroscopy c) immunological analysis; d)production of the corresponding RNA; or e) production of thecorresponding protein.
 16. The method of claim 10, wherein the extractedRNA is mammalian RNA.
 17. A method for detecting tumor-associated ortumor-derived RNA in a non-cellular fraction of a bodily fluid from ahuman or animal, wherein the tumor-associated or tumor-derived RNA isRNA of a non-mutated gene, the method comprising the steps of: a)extracting total RNA from a non-cellular fraction of a bodily fluid froma human or animal, wherein a fraction of said extracted RNA comprises anRNA species of a non-mutated gene; b) amplifying or signal amplifyingthe non-mutated mRNA or a cDNA produced therefrom to produce anamplified fragment or amplified signal, wherein amplification isperformed qualitatively or quantitatively using primers or probesspecific for the tumor-derived or tumor-associated, non-mutated mRNA orcDNA; and c) detecting quantitatively or qualitatively the amplifiedfragment or amplified signal produced from the tumor-derived ortumor-associated, non-mutated mRNA or cDNA.
 18. The method of claim 17,wherein the extracted RNA is mammalian RNA.
 19. A method for detectingRNA in blood plasma or serum from a human or animal, wherein the RNA isa species that is detected in blood plasma or serum more frequently whenthe blood plasma or serum is obtained from an individual with cancerthan when it is obtained from an individual without cancer, the methodcomprising the steps of: a) extracting total RNA from plasma or serum ofa human or animal, wherein a fraction of said extracted RNA comprises anRNA species that is detected in blood plasma or serum more frequentlywhen the blood plasma or serum is obtained from an individual withcancer than when it is obtained from an individual without cancer; b)amplifying or signal amplifying said species of RNA or cDNA preparedtherefrom that is detected in blood plasma or serum more frequently whenthe blood plasma or serum is obtained from an individual with cancerthan when it is obtained from an individual without cancer, to producean amplified fragment or amplified signal, wherein amplification isperformed qualitatively or quantitatively using primers or probesspecific for the RNA or cDNA; and c) detecting quantitatively orqualitatively the amplified fragment or amplified signal produced fromthe RNA or cDNA species.
 20. The method of claim 19, wherein RNA or cDNAproduced therefrom is amplified using a method that is: a) reversetranscriptase polymerase chain reaction; b) ligase chain reaction; c)branched DNA signal amplification; d) amplifiable RNA reporters; e)Q-beta replication; f) transcription-based amplification; g) isothermalnucleic acid sequence replication assay; h) boomerang DNA amplification;i) strand displacement activation; j) cycling probe technology; or k) acombination or variation thereof that amplifies the RNA or cDNA.
 21. Themethod of claim 19, wherein the amplified fragment is detected using amethod that is: a) an electrophoresis-based detection method b) ELISAdetection methods using biotinylated or other modified primers; c)immunological detection methods using monoclonal antibodies; d)detection methods using a labeled fluorescent or chromagenic probe; e)Southern blot analysis f) electrochemiluninescence g) reverse dot blotdetection; or h) high-performance liquid chromatography.
 22. The methodaccording to claim 19, wherein the amplified fragment produced accordingto step b) is further characterized by: a) nucleic acid sequencing b)spectroscopy c) immunological analysis d) production of thecorresponding RNA; or e) production of the corresponding protein. 23.The method of claim 19, wherein amplified fragment produced in step b)is further characterized by: a) nucleic acid sequencing; b)spectroscopy; c) immunological analysis; d) production of acorresponding RNA; or e) production of a corresponding protein.
 24. Themethod of claim 19, wherein the extracted RNA is mammalian RNA.
 25. Amethod for detecting a plurality of RNA species in plasma or serum froma human or animal, the method comprising the steps of: a) extractingtotal RNA from plasma or serum of a human or animal, wherein saidextracted RNA comprises a plurality of mammalian RNA species; b)amplifying or signal amplifying concurrently or sequentially at leasttwo of said plurality of RNA or cDNA produced therefrom to therebyproduce amplified fragments or amplified signals, wherein amplificationis performed qualitatively or quantitatively using at least two primersor probes, each specific for an RNA species or cDNA produced therefrom;and c) detecting the amplified fragments or amplified signals producedfrom at least two of the amplified RNA species or cDNA producedtherefrom.
 26. The method of claim 25, wherein one or more RNA speciesis a tumor-associated or tumor-derived RNA.
 27. The method of claim 25,wherein RNA or cDNA produced therefrom is amplified using a method thatis: a) reverse transcriptase polymerase chain reaction; b) ligase chainreaction; c) branched DNA signal amplification; d) amplifiable RNAreporters; e) Q-beta replication; f) transcription-based amplification;g) isothermal nucleic acid sequence replication assay; h) boomerang DNAamplification; i) strand displacement activation; j) cycling probetechnology: or k) a combination or variation thereof that amplifies theRNA or cDNA.
 28. The method of claim 25, wherein the amplified fragmentis detected using a method that is: a) an electrophoresis-baseddetection method; b) ELISA detection methods using biotinylated or othermodified primers; c) immunological detection methods using monoclonalantibodies; d) detection methods using a labeled fluorescent orchromagenic probe; e) Southern blot analysis; f)electrochemiluminescence g) reverse dot blot detection; or h)high-performance liquid chromatography.
 29. The method of claim 25,wherein at least one of the RNA species is tyrosinase RNA, keratin RNA,prostate-specific antigen RNA, alpha-fetoprotein RNA, BCR/abl RNA,carcinoembryonic antigen RNA, p97 RNA, MUC 18 RNA, PML/RAR RNA, CD44RNA, EWS/FLI-1 RNA, EWS/ERG RNA, AML1/ETO RNA, MAGE RNA, beta humanchorionic gonadotropin RNA, or telomerase-associated RNA.
 30. The methodof claim 25, wherein at least one RNA species is from a non-mutatedgene.
 31. The method of claim 25, wherein the extracted RNA is mammalianRNA.
 32. A method for diagnosing, detecting or monitoring a disease in ahuman or animal, the method comprising the step of detecting at leasttwo of a plurality of RNA species that are expressed in humans oranimals having the disease, or cDNA produced therefrom, according to themethod of claim 25, whereby a disease is diagnosed, detected, ormonitored.
 33. The method of claim 32, wherein the disease is cancer orpremalignancy.
 34. The method of claim 32, wherein the extracted RNA ismammalian RNA.
 35. A method for detecting a combination of RNA speciesin blood plasma or serum from a human or animal, wherein detection ofsaid combination indicates a disease in the animal or human, the methodcomprising the steps of: a) extracting total RNA from plasma or serumfrom a human or animal, wherein said extracted RNA comprises a pluralityof mammalian RNA species; b) amplifying or signal amplifyingconcurrently or sequentially two or more of said plurality of RNA orcDNA produced therefrom to thereby produce amplified fragments oramplified signals, wherein amplification is performed qualitatively orquantitatively using at least two primers or probes, each specific forRNA species comprising parts of a combination of RNA species indicativeof a disease, or cDNA thereof; and c) qualitatively or quantitativelydetecting at least two of the amplified fragments or amplified signalsproduced from the combination of RNA species or cDNAs preparedtherefrom.
 36. The method of claim 35, wherein one or more RNA speciesin said combination are tumor-associated or tumor-derived RNA.
 37. Themethod of claim 35, wherein at least one RNA species in said combinationis tyrosinase RNA, keratin RNA, prostate-specific antigen RNA,alpha-fetoprotein RNA, BCR/abl RNA, carcinoembryonic antigen RNA, p97RNA, MUC 18 RNA, PML/RAR RNA, CD44 RNA, EWS/FLI-1 RNA, EWS/ERG RNA,AML/ETO RNA, MAGE RNA, beta human chorionic gonadotropin RNA, ortelomerase-associated RNA.
 38. The method of claim 35, wherein thedisease is cancer or premalignancy.
 39. The method of claim 35, whereinthe extracted RNA is mammalian RNA.